Fig. 2. Evaluation of the specificity of the duplex real-time PCR assay with internal standard. Real-time PCR amplifications of plasma DNA, internal standard DNA, and the mixture of both in a parallel PCR reaction with their corresponding primers and probes are performed in preliminary experiments to confirm the specificity of this novel duplex PCR assay (A). The target β–actin gene and internal standard are amplified by their corresponding primers for 91-bp and 99-bp PCR products respectively (B) and Taqman probes for fluorescent signals released from JOE and FAM reports and detected in channels 2 and 1 of the ABI 7500 Sequence Detector (C).